COMPOSITIONS AND METHODS FOR cDNA SYNTHESIS

ABSTRACT

Methods for making cDNA molecules, for amplification of RNA by PCR and for preparation of cDNA libraries are provided. Kits for making cDNA molecules also are provided. 
     Compositions are also provided comprising mixtures of reagents, including reverse transcriptases, buffers, cofactors and other components, suitable for immediate use in conversion of RNA into cDNA and RT PCR without dilution or addition of further components. These compositions are useful, alone or in the form of kits, for cDNA synthesis or nucleic acid amplification (e.g., by the Polymerase Chain Reaction) or for any procedure utilizing reverse transcriptases in a variety of research, medical, diagnostic, forensic and agricultural applications.

The application is a continuation of U.S. patent application Ser. No.12/632,802 filed Dec. 8, 2009, which is a continuation of U.S. patentapplication Ser. No. 10/653,416 filed Sep. 3, 2003, now U.S. Pat. No.7,638,612, which claims the benefit of U.S. Provisional Application Ser.No. 60/407,248, filed Sep. 3, 2002, the contents of each of which areincorporated herein by reference in their entireties. This applicationalso contains a sequence listing submitted electronically via EFS-web,which serves as both the paper copy and the computer readable form (CRF)and consists of a file entitled “01230_(—)8003_US04_SEQ_LIST”, which wascreated on Mar. 4, 2013, which is 5,471 bytes in size, and which isherein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention provides methods and compositions for preparing cDNA

BACKGROUND OF THE INVENTION

In examining the structure and physiology of an organism, tissue orcell, it often is desirable to determine its genetic content. Thegenetic framework of an organism is encoded in the double-strandedsequence of nucleotide bases in the deoxyribonucleic acid (DNA) which iscontained in the somatic and germ cells of the organism. The geneticcontent of a particular segment of DNA, or gene, is manifested only uponproduction of the protein encoded by the gene. To produce a protein, acomplementary copy of one strand of the DNA double helix (the “coding”strand) is produced by polymerase enzymes, resulting in a specificsequence of ribonucleic acid (RNA). This particular type of RNA, sinceit contains the genetic message from the DNA for production of aprotein, is called messenger RNA (mRNA).

Within a given cell, tissue or organism, there exist many mRNA species,each encoding a separate and specific protein. This fact provides apowerful tool to investigators interested in studying genetic expressionin a tissue or cell. mRNA molecules may be isolated and furthermanipulated by various molecular biological techniques, thereby allowingthe elucidation of the full functional genetic content of a cell, tissueor organism. The identity and levels of specific mRNAs present in aparticular sample provides clues to the biology of the particular tissueor sample being studied. Therefore, the detection, analysis,transcription, and amplification of RNAs are among the most importantprocedures in modem molecular biology.

A common approach to the study of gene expression is the production ofcomplementary DNA (cDNA). In this technique, the mRNA molecules from anorganism are isolated from an extract of the cells or tissues of theorganism. From these purified mRNA molecules, cDNA copies may be madeusing the enzyme reverse transcriptase (RT) or DNA polymerases having RTactivity, which results in the production of single-stranded cDNAmolecules. The term “reverse transcriptase” describes a class ofpolymerases characterized as RNA dependent DNA polymerases. All knownreverse transcriptases require a primer to synthesize a DNA transcriptfrom an RNA template.

Avian myoblastosis virus (AMV) reverse transcriptase was the firstwidely used RNA dependent DNA polymerase (Verma, Biochem. Biophys. Acta473:1 (1977)). The enzyme has 5′-3′ RNA directed DNA polymeraseactivity, 5′-3′ DNA directed DNA polymerase activity, and RNase Hactivity. RNase H is a processive 5′ and 3′ ribonuclease specific forthe RNA strand for RNA DNA hybrids (Perbal, A Practical Guide toMolecular Cloning, New York: Wiley & Sons (1984)). Errors intranscription cannot be corrected by reverse transcriptase because knownviral reverse transcriptases lack the 3′-5′ exonuclease activitynecessary for proofreading (Saunders and Saunders, Microbial GeneticsApplied to Biotechnology, London: Croom Helm (1987)). A detailed studyof the activity of AMV reverse transcriptase and its associated RNase Hactivity has been presented by Berger et al., Biochemistry 22:2365 2372(1983).

Another reverse transcriptase which is used extensively in molecularbiology is reverse transcriptase originating from Moloney murineleukemia virus (M-MLV). See, e.g., Gerard, G. R., DNA 5:271 279 (1986)and Kotewicz, M. L., et al., Gene 35:249 258 (1985). M-MLV reversetranscriptase substantially lacking in RNase H activity has also beendescribed. See, e.g., U.S. Pat. No. 5,244,797.

Historically, reverse transcriptase has been used primarily totranscribe mRNA into cDNA which can then be cloned into a vector forfurther manipulation. The single-stranded cDNAs may be converted into acomplete double-stranded DNA copy (i.e., a double-stranded cDNA) of theoriginal mRNA (and thus of the original double-stranded DNA sequence,encoding this mRNA, contained in the genome of the organism) by theaction of a DNA polymerase. The double stranded cDNAs can then beinserted into a vector, transformed into an appropriate bacterial,yeast, animal or plant cell host, and propagated as a population of hostcells containing a collection of cDNA clones, or cDNA library, thatrepresents the genes, or portions of genes present in the original mRNAsample.

Alternatively, cDNA can be labeled with an appropriate reporter moietyand used as hybridization probe to query defined target sequencesimmobilized on glass slides, filters, or other suitable solid supports.The identity and relative abundance of a given mRNA in a sample can beinferred from the signal intensity for a specific target sequence on thesolid support.

One of the most widely used techniques to study gene expression exploitsfirst-strand cDNA for mRNA sequence(s) as template for amplification bythe polymerase chain reaction, PCR. This method, often referred to asRNA PCR or reverse transcriptase PCR (RT-PCR), exploits the highsensitivity and specificity of the PCR process and is widely used fordetection and quantification of RNA. Recently, the ability to measurethe kinetics of a PCR reaction by on-line detection in combination withthese RT-PCR techniques has enabled accurate and precise measurement ofRNA sequences with high sensitivity. This has become possible bydetecting the RT-PCR product through fluorescence monitoring andmeasurement of PCR product during the amplification process byfluorescent dual-labeled hybridization probe technologies, such as the“TaqMan” 5′ fluorogenic nuclease assay described by Holland et al.(Proc. Natl. Acad. Sci. U.S.A. 88, 7276 (1991)), Gibson et al. (GenomeRes. 6, 99 (1996)), and Heid et al. (Genome Res. 6, 986 (1996)); or“Molecular Beacons” (Tyagi, S. and Kramer, F. R. Nature Biotechnology14, 303 (1996)). Nazarenko et al. (Nucleic. Acids Res. 25, 2516 (1997))have described use of dual-labeled hairpin primers, as well as recentmodifications utilizing primers labeled with only a single fluorophore(Nazerenko et al., Nucleic. Acids Res. (2002)). One of the more widelyused methods is the addition of double-strand DNA-specific fluorescentdyes to the reaction such as: ethidium bromide (Higuchi et al.,Biotechnology (1992) and Higuchi et al., Biotechnology 11, 102610, 413(1993)), YO-PRO-1 (Ishiguro et al., Anal. Biochem. 229, 207 (1995)), orSYBR Green I (Wittwer et al., Biotechniques 22,130 (1997)). Theseimprovements in the PCR method have enabled simultaneous amplificationand homogeneous detection of the amplified nucleic acid withoutpurification of PCR product or separation by gel electrophoresis. Thiscombined approach decreases sample handling, saves time, and greatlyreduces the risk of product contamination for subsequent reactions, asthere is no need to remove the samples from their closed containers forfurther analysis. The concept of combining amplification with productanalysis has become known as “real time” PCR, also referred to asquantitative PCR, or qPCR.

The general principals for template quantification by real-time PCR werefirst disclosed by Higuchi R, G Dollinger, P S Walsh and R. Griffith,“Simultaneous amplification and detection of specific DNA sequences”,Bio/Technology 10:413-417, 1992; Higuchi R, C Fockler G Dollinger and RWatson, Kinetic PCR analysis: real time monitoring of DNA amplificationreactions, Bio/Technology 11:1026-1030. This simpler approach forquantitative PCR utilizes a double-strand specific fluorescent dye,ethidium bromide, added to amplification reaction. The fluorescentsignal generated at each cycle of PCR is proportional to the amount ofPCR product. A plot of fluorescence versus cycle number is used todescribe the kinetics of amplification and a fluorescence thresholdlevel was used to define a fractional cycle number related to initialtemplate concentration. Specifically, the log of the initial templateconcentration is inversely proportional to the fractional cycle number(threshold cycle, or Ct), defined as the intersection of thefluorescence versus cycle number curve with the fluorescence threshold.Higher amounts of starting template results in PCR detection at a lowerCt value, whereas lower amounts require a greater number of PCR cyclesto achieve an equivalent fluorescent threshold (Ct) and are detected athigher Ct values. Typically, the setting of this fluorescence thresholdis defined as a level that represents a statistically significantincrease over background fluorescent noise. Since this occurs at anearly stage in the PCR process when critical substrates are notlimiting, quantification of starting template occurs over a broaddynamic range with high accuracy, precision, and sensitivity. A majorproblem in understanding of gene expression patterns for gene discoveryand identification of metabolic pathways is the limitations of currentmethods for accurate quantification. Use of real time PCR methodsprovides a significant improvement towards this goal. However, real-timePCR quantification of mRNA is still bounded by limitations of theprocess of reverse transcription.

The RT-PCR procedure, carried out as either an end-point or real-timeassay, involves two separate molecular syntheses: (i) the synthesis ofcDNA from an RNA template; and (ii) the replication of the newlysynthesized cDNA through PCR amplification. To attempt to address thetechnical problems often associated with RT-PCR, a number of protocolshave been developed taking into account the three basic steps of theprocedure: (a) the denaturation of RNA and the hybridization of reverseprimer; (b) the synthesis of cDNA; and (c) PCR amplification. In the socalled “uncoupled” RT-PCR procedure (e.g., two step RT-PCR), reversetranscription is performed as an independent step using the optimalbuffer condition for reverse transcriptase activity. Following cDNAsynthesis, the reaction is diluted to decrease MgCl₂, anddeoxyribonucleoside triphosphate (dNTP) concentrations to conditionsoptimal for Taq DNA Polymerase activity, and PCR is carried outaccording to standard conditions (see U.S. Pat. Nos. 4,683,195 and4,683,202). By contrast, “coupled” RT PCR methods use a common orcompromised buffer for reverse transcriptase and Taq DNA Polymeraseactivities. In one version, the annealing of reverse primer is aseparate step preceding the addition of enzymes, which are then added tothe single reaction vessel. In another version, the reversetranscriptase activity is a component of the thermostable Tth DNApolymerase. Annealing and cDNA synthesis are performed in the presenceof Mn⁺⁺ then PCR is carried out in the presence of Mg⁺⁺ after theremoval of Mn⁺⁺ by a chelating agent. Finally, the “continuous” method(e.g., one step RT-PCR) integrates the three RT-PCR steps into a singlecontinuous reaction that avoids the opening of the reaction tube forcomponent or enzyme addition. Continuous RT-PCR has been described as asingle enzyme system using the reverse transcriptase activity ofthermostable Taq DNA Polymerase and Tth polymerase and as a two enzymesystem using AMV RT and Taq DNA Polymerase wherein the initial 65° C.RNA denaturation step was omitted.

One step RT-PCR provides several advantages over uncoupled RT-PCR. Onestep RT-PCR requires less handling of the reaction mixture reagents andnucleic acid products than uncoupled RT-PCR (e.g., opening of thereaction tube for component or enzyme addition in between the tworeaction steps), and is therefore less labor intensive, reducing therequired number of person hours. One step RT-PCR also requires lesssample, and reduces the risk of contamination (Sellner and Turbett,1998). The sensitivity and specificity of one-step RT-PCR has provenwell suited for studying expression levels of one to several genes in agiven sample or the detection of pathogen RNA. Typically, this procedurehas been limited to use of gene-specific primers to initiate cDNAsynthesis.

In contrast, use of non-specific primer in the “uncoupled” RT-PCRprocedure provides opportunity to capture all RNA sequences in a sampleinto first-strand cDNA, thus enabling the profiling and quantitativemeasurement of many different sequences in a sample, each by a separatePCR. The ability to increase the total amount of cDNA produced, and moreparticularly to produce cDNA that truly represents the mRNA populationof the sample would provide a significant advance in study of geneexpression. Specifically, such advances would greatly improve theprobability of identifying genes which are responsible for disease invarious tissues.

Ideally, synthesis of a cDNA molecule initiates at or near the3′-termini of the mRNA molecules and terminates at the mRNA 5′-end,thereby generating “full-length” cDNA. Priming of cDNA synthesis at the3′-termini at the poly A tail using an oligo dT primer ensures that the3′-message of the mRNAs will be represented in the cDNA moleculesproduced. It would be very desirable if cDNA synthesis initiated at 3′end and continued to the 5′-end of mRNA's regardless of length of mRNAand the reverse transcriptase used. However, due to many factors such aslength, nucleotide sequence composition, secondary structure of mRNA andalso inadequate processivity of reverse transcriptases, cDNA synthesisprematurely terminates resulting in non-quantitative representation ofdifferent regions of mRNA (i.e. 3′-end sequences or 5′-end sequences).It has been demonstrated that use of mutant reverse transcriptaseslacking RNase H activity result in longer cDNA synthesis and betterrepresentation, and higher sensitivity of detection. However, it isgenerally believed that using oligo dT primer results in cDNA sequencebias of mRNA 3′-end region.

In studies involving quantitative analysis of gene expression, sequencebias in the cDNA and non-quantitative representation of different partsof mRNA can yield inaccurate expression data. Due to these problems analternative method of priming for cDNA synthesis has been used utilizingrandom primers. Due to random sequence, these primers are believed tonon-specifically prime cDNA synthesis at arbitrary sites along the mRNAresulting shorter cDNA fragments that collectively represent all partsof mRNA in the cDNA population. Gerard and D'Alessio (1993 Methods inMolecular Biology 16:73-93) have reported that the ratio of randomprimer to mRNA is critical for efficient cDNA synthesis by M-MLV RT orits RNase H deficient derivatives. Increasing concentrations of randomhexamer resulted in increased yields of cDNA, however the average lengthof cDNA decreased accordingly. At equal hexamer concentrations, use ofRNase IT RT resulted in cDNA yields that were approximately 4 foldhigher than that obtained with M-MLV RT. Ratios of hexamer to mRNA of10:1 for M-MLV H-RT and 40:1 for M-MLV RT were reported to producereasonable yields of cDNA without sacrificing length. This indicatesthat primer concentration must be optimized for different amounts ofstarting RNA template to achieve efficient cDNA synthesis efficiency.Since random primer has the potential to omit sequence close to the mRNApolyA tail, in some protocols, oligo dT primer and random primers havebeen used as mixtures and combine both priming methods.

The choice and concentration of primer can have a profound impact on thequantitative representation of different mRNA transcripts infirst-strand cDNA. It is apparent therefore, that improved compositionsand methods for improving the yield of cDNA produced using reversetranscription are greatly to be desired. It is also apparent that newmethods for making collections or libraries of cDNA from cells or tissuethat more accurately represent the relative amounts of mRNAs present inthe cells or tissue are greatly to be desired. It is also apparent thatmore convenient compositions and kits for use in such methods aredesirable.

SUMMARY OF THE INVENTION

The instant invention provides improved methods of synthesizing a cDNAmolecule or molecules from an mRNA template or population of mRNAtemplates under conditions sufficient to increase the total amount ofcDNA produced, and/or reduce RNA sequence bias in the resulting cDNAproduct. Specifically, the invention relates to the use of a mixture ofoligo(dT) primer and random primer (e.g. hexameric, heptameric,octameric, nonameric, etc.) in a first-strand cDNA synthesis reaction.In accordance with the invention, any conditions that improve primingmay be used. Such conditions preferably include, but are not limited to,optimizing primer concentrations, optimizing reaction temperaturesand/or optimizing primer length or specificity. Such results may also beaccomplished in accordance with the invention by optimizing the reversetranscription reaction, preferably by balancing the composition of saltsor including enhancing agents that disrupt RNA secondary structure orimprove the processivity of reverse transcriptase.

The present invention is also directed to compositions comprisingmixtures of reagents, including reverse transcriptases, buffers,cofactors and other components, suitable for immediate use in conversionof RNA into cDNA and RT PCR without dilution or addition of furthercomponents. These compositions are useful, alone or in the form of kits,for cDNA synthesis or nucleic acid amplification (e.g., by thePolymerase Chain Reaction) or for any procedure utilizing reversetranscriptases in a variety of research, medical, diagnostic, forensicand agricultural applications.

It is therefore an object of this invention to provide new methods forreverse transcription of one or more nucleic acid molecules comprisingincubating one or more nucleic acid templates in a buffer underconditions sufficient to make one or more first nucleic acid moleculescomplementary to all or a portion of the one or more templates, wherethe buffer comprises at least one reverse transcriptase, an effectiveamount of a mixture of random primers, where the random primers arepresent in a concentration of at least about 5 ng/μl, and an effectiveamount of oligo(dT), where the oligo(dT) is present in a concentrationless than about 2 μM.

In accordance with further aspects of the invention the random primersmay be present in a concentration of between about 5 ng/μl and about 20ng/μl. The oligo(dT) may be present in a concentration of between about25 nM and about 2 μM. The random primers may be between 5 and 10nucleotides long, and may be are random hexamers. The oligo(dT) mayconsist essentially of between about 12 and about 25 dT residues, andmay be an anchored oligo(dT) molecule containing a terminal non-Tnucleotide or dinucleotide. The oligo(dT) may oligo(dT)₁₂₋₁₈ oroligo(dT)₂₀ or anchored equivalents thereof.

In another embodiment the reverse transcriptase may be a viral reversetranscriptase, and may be selected from the group consisting of AMV RT,RSV RT, MMLV RT, HIV RT, EIAV RT, RAV2 RT, TTH DNA polymerase, C.hydrogenoformans DNA polymerase, Superscript II RT, Superscript I RT,Thermoscript RT MMLV, ASLV and Rnase H⁻ mutants thereof. Mixtures of anyof these reverse transcriptases may be used. In particular, mixtures ofviral RT enzymes may be used, such as mixtures of MMLV and ASLV, and/ortheir RNAse H reduced or RNAse H⁻ analogs may be used.

It is another object of the invention to provide methods for reversetranscription of one or more nucleic acid molecules comprisingincubating one or more nucleic acid templates in a buffer underconditions sufficient to make one or more first nucleic acid moleculescomplementary to all or a portion of the one or more templates, wherethe buffer comprises at least one reverse transcriptase, one or moreprimers suitable for priming reverse transcription of the one or moretemplates; and an effective amount of Li ion.

In accordance with further aspects of the invention the reversetranscriptase may be a viral reverse transcriptase, and may be selectedfrom the group consisting of AMV RT, RSV RT, MMLV RT, HIV RT, EIAV RT,RAV2 RT, Superscript II RT, Superscript I RT, Thermoscript RT MMLV andRnase H⁻ mutants thereof.

In one aspect of the invention, the Li ion may be present in aconcentration of between about 5 mM to about 200 mM. The buffer mayfurther comprise at least one additional monovalent cation in aconcentration between about 20 mM and 200 mM, where the monovalentcation is selected from the group consisting of Na, K, and NH4, andwhere the total concentration of the Li ion and the further monovalentcation is less than or equal to about 200 mM. The additional monovalentcation may be K.

It is another object of the invention to provide a reagent mixturesuitable for use in a reverse transcription reaction of at least onetemplate nucleic acid, comprising glycerol in a concentration betweenabout 10% and about 40%, a buffer and a reverse transcriptase, where thereagent mixture demonstrates prolonged stability when stored at −20° C.and may be used directly for a reverse transcription reaction withoutadding additional reverse transcriptase.

In one aspect of the invention, the buffer may comprise a monovalentcation selected from the group consisting of Li, Na, K and NH₄, amagnesium salt, a reducing agent, nucleoside triphosphates, and at leastone non-ionic detergent. The buffer may further comprise at least oneprimer suitable for priming reverse transcription of a template by thereverse transcriptase. The mixture may also comprise an RNAse inhibitorprotein. In one embodiment, the buffer comprises a potassium salt, amagnesium salt, nucleoside triphosphates, DTT, at least one primersuitable for priming reverse transcription of a template by the reversetranscriptase, at least one non-ionic detergent, and an RNAse inhibitorprotein.

In any of these methods and compositions, two or more reversetranscriptases may be used, including any reverse transcriptase asdescribed above. In any of these methods and compositions at least onethermostable DNA polymerase may also be present.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows sequences of qPCR Primers and Target mRNA SequenceInformation in Table 1.

FIG. 2 shows average C_(T) values for SYBR Green qRT-PCR of first-strandcDNAs primed with varying amounts of random hexamer or oligo(dT)20 inTable 2.

FIG. 3 shows average C_(T) values for SYBR Green qRT-PCR of first-strandcDNAs synthesized in the presence of different cations in Table 3. Boldtext indicates lowest average C_(T) for each reaction set. (NA=noamplification)

FIG. 4 shows stability information for various reaction mixtures for usein reverse transcription reactions Tables 4A, 4B and 4C.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, it has been have found that by varying the concentrationand ratios of oligo dT and random primers, efficiency of cDNA synthesisand uniform representation of mRNA sequences can be vastly improved.These improvements were realized using a fixed quantity of an oligo(dT)and random primer mixture over a wide range of starting RNA templateamounts. Even though the ratio of primer to mRNA varied over 6 orders ofmagnitude, both the relative and absolute representation of mRNAsequence was maintained in the cDNA. In contrast to findings of earlierstudies, it has been surprisingly discovered that even when using wildtype reverse transcriptases with full RNase H activity, sensitivity ofdetection can be improved due to better and more efficient conversion ofmRNA into cDNA.

An additional surprising discovery is that improved reaction conditionsfor cDNA synthesis can be obtained through the inclusion of an effectiveamount of a lithium salt in the reaction mixture, resulting inunexpectedly increased cDNA yield, particularly at low RNA templateconcentrations. Other embodiments of the invention relate to stabilizedconcentrated reaction mixtures for first-strand cDNA synthesis thatsimplify and improve the reliability of reverse transcription.

The present invention therefore relates to methods of increasing theefficiency of cDNA synthesis and more particularly, to increasing thesensitivity and accuracy of quantification of gene expression. Thus, thepresent invention provides improved cDNA synthesis useful in genediscovery, genomic research, diagnostics and identification ofdifferentially expressed genes and identification of genes of importanceto disease.

Use of Primer Combinations

Specifically, the present invention describes new primer combinationsthat provide more efficient and uniform priming for cDNA synthesis. Theconcentration and combinations of random primers and oligo dT usedprovides efficient and representative conversion of mRNA sequences intocDNA. This method provides superior and non-biased conversion of mRNAsequences into cDNA regardless of distance from 3′ end of mRNA.

In one embodiment of this invention the random primers are mixed witholigo dT for priming cDNA synthesis. A variety of concentrations andratios of each primer type can be used according to the method of theinvention.

Surprisingly it has been found that optimal amplification of some genesrequires oligo dT while others require random primers. By combining bothprimer types as a mix this invention provides optimal cDNA synthesis andamplification for all mRNAs regardless of proximity of amplificationregion to 3′ or 5′ ends. The random primers used according to theinvention can vary in size from 5 bases to 12 bases.

The length of oligo dT can vary from 8 bases to 30 bases. Other types ofprimers with different composition can be used in place of oligo dT.Examples of such compositions include, but are not limited to, oligo dTwhere the 3′ base is A, or C, or G (anchored dT). Alternatively, twobases at the 3′ end can be variable and can be any combination of A, C,or G. Other sequences or moieties that can base pair with poly Asequences of mRNA can also be used. An example, without limitation, isdeoxy uridine, (dU).

The amount of random primers can vary from 25 ng to 800 ng for eachreaction (20 uL), for example, 25, 50, 75, 100, 200, 300, 400, 500, 600,or 700 ng, or intermediate values. According to the methods of theinvention the concentration of oligo dT can be 25 nM to 5000 nM, forexample for example, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 2000, 3000, or 4000 nM, or intermediate values. It willbecome evident to those skilled in the art that various ratios of randomprimers and oligo dT can be used.

The skilled artisan will recognize that when concentrations of randomprimers or oligo dT are specified as weight/volume ratios, the reagentconcentrations obtained using such quantities will vary depending on thelength of the primer and the attendant change in molecular weight. Forexample, the skilled worker will know that, when a method employs 12.5ng/ml of random hexamers, an adjustment of quantity is required toachieve an equivalent concentration when random nonamers are used.Similar adjustments are made when using different lengths of oligo dTprimers, such as oligo(dT)₂₀ and oligo dT)₁₂₋₁₈.

Use of Lithium Ion in RT and RT-PCR Reactions

In other embodiments of the present invention, it has been found thatwhen lithium-containing compounds are included in the cDNA reaction theefficiency of cDNA synthesis is improved, resulting in highersensitivity of detection and more accurate quantitation (see Examples).A variety of lithium containing salts and compounds can be used, and theskilled artisan will appreciate that the identity and concentration ofthe salt can be varied to optimize results. In the context of thepresent invention, an effective amount of a lithium salt is an amountsufficient to permit RT activity, or in the case of RT reactions thatcontain an additional monovalent cation such as K, that produce improvedcDNA synthesis compared to the results produced in the absence of Li.

It is surprising that lithium can be used at all in RT reactions becauseit previously was thought that Li ion was inhibitory for a variety ofretroviral enzymes and lithium is known to inhibit viral replication.The fact that lithium not only does not inhibit the reaction butproduces improved results is particularly surprising.

Convenient and Stable Reagent Compositions

Another embodiment of the present invention is the form in which thereaction mixture is prepared and stably maintained. Traditionally, cDNAreaction components have been supplied as a number of separatecomponents that are assembled into a complete reaction mix just prior tostart of cDNA synthesis. Indeed, there is a widespread prejudice in theart that these components need to be kept separate for storage purposes.A typical kit for cDNA synthesis contains the following components:

-   a. Oligo(dT) 12-18. 50 μl of a 0.5 mg/ml solution.-   b. Random hexamers. 50 μl of a 50 ng/μl solution. This is a 25 μM    solution.-   c. 10×RT Buffer. 1 ml of solution containing 200 mM TrisCl pH 8.4,    500 mM KCl.-   d. 25 mM MgCl₂. 500 μl supplied.-   e. 0.1 M DTT. 250 μl.-   f. dNTP mix. 250 μl of a solution containing 10 mM each of dATP,    dCTP, dGTP, dTTP.-   g. SUPERSCRIPT II RT. 50 μl of enzyme at 50 U/μl.-   h. E. coli RNase H. 50 μl of enzyme at 2 U/μl.-   i. DEPC-treated water. 1.2 ml.-   j. RNASEOUT™ Recombinant Ribonuclease Inhibitor. 100 μl supplied at    40 units/μl.

Each of the above components are provided separately and are frozen at−20° C. for storage. The above components are the components ofSuperScript® 1^(st) strand synthesis system for RT PCR from InvitrogenCorporation and is provided as a typical example of cDNA synthesis kits.All other commercially available kits are very similar. The generalbelief has been that the components can not be mixed for long termstorage. A key component of these systems is reverse transcriptase thatis always stored in special storage buffer with at least 50% glycerol,and is only added to the reaction mix immediately prior to start of cDNAsynthesis.

Surprisingly, we have found that some or all of the components of thecDNA synthesis reaction can be combined and stored as a convenientready-to-use mix that is stable to prolonged storage at −20° C. and thatcan simply be added to a nucleic acid template solution when needed. Theready to use reaction mixture may contain between about 10 and 40%glycerol, which is significantly less than the 50% or more thatpreviously was thought necessary to maintain stability of the RT enzymethat is present in the mix.

The following is a formulation for a 5× cDNA mastermix that hassuccessfully been produced and used for a variety of applications. Theminimum components that may usefully be provided for the mixture are theglycerol, the RT and a suitable buffer component. Suitable buffercompounds, such as Tris-HCl, HEPES, etc, are well known in the art.Metal ions necessary for RT activity, such as Mg and a monovalent cationsuch as Li, K, Na, or NH₄ may be present in concentrations that aresuitable for RT activity upon addition to a template solution.Additional components that may be present are a reducing agent, such asDTT, primer molecules such as gene specific primers, random primers ofany suitable length, oligo(dT) compounds of any suitable length,anchored oligo(dT) molecules of suitable length, detergents or mixturesof detergents such as Tween, NP-40 and Igepal CA-630 and equivalentreagents, dNTPs, and one or more RNAse inhibitor proteins. The relativeamounts contained in the mixture of such reagents necessary for use inRT reactions, when present, can be readily determined by the skilledartisan. In addition, at least one thermostable DNA polymerase may alsobe present, which may be used for subsequent PCR reactions or the like.

Accordingly, the present invention provides newly improved, convenient,and ready to use configurations for cDNA synthesis. The methods of theinvention reduce the number additions for assembly of cDNA synthesisreactions which is highly sought by researchers especially in highthroughput applications.

According to the methods of the invention, the ready to use mixes forcDNA synthesis can be made at different concentrations and provided as1× to 20×“mastermixes.” The following is an example of a 5× mastermixfor cDNA synthesis that contains all components necessary for cDNAsynthesis according to the methods of this invention. Using 4 uL of thismastermix and RNA preparation of interest at a total volume of 20 uLprovides a complete reaction mix for conversion of RNA into cDNA. Theskilled artisan will readily appreciate how to prepare suitable 1× to20× mastermixes.

Formulation for 5× cDNA mastermix:

5× Buffer (0.1 M Tris-HCl, pH 8.4, 0.25M KCl) 0.1 M LiCl 25 mM MgCl2

2.5 mM dNTP (each)

50 mM DTT

500 nM oligo(dT)2050 ug/mL random primer

30% Glycerol 0.005% Igepal CA-630 0.005% Tween 20 10,000 U/mL MMLV RT

5000 U/mL RNase inhibitor protein

In addition to the above formulation, three other mastermixes wereprepared that contained all reagents except the primers.

RT Mix 1 did not have primers

RT Mix 2 contained oligo dT as the primers

RT Mix 3 contained Random hexamers and Octamers as primers.

All of the above 5× cDNA mastermixes were found to be stable for monthswhen stored at −20 C. Table 4 shows the results and the efficacy of cDNAsynthesis with these mastermixes compared to the reagents storedseparately under the conditions recommended in the literature.

It will be evident to those skilled in the art that a variety ofdifferent reverse transcriptases can be used according to the method ofthe invention. The reverse transcriptases may include, withoutlimitation, AMV RT, RSV RT, MMLV RT, Rnase H-mutants of various reversetranscriptases, HIV RT, EIAV RT, RAV2 RT, TTH DNA polymerase, C.hydrogenoformans DNA polymerase, Superscript II RT, Superscript I RT,Thermoscript RT and mixtures thereof. It will also be obvious that oneor more of the components of the above mastermix can be substituted withother equivalent reagent or protein. For example, there are a number ofdifferent RNAse inhibitor proteins that can be used. If desired, theRNAse inhibitor protein can also be excluded from the mixture since itis not always necessary for cDNA synthesis. Thermostable DNA polymerasessuitable for use in the mastermixes are well known in the art andinclude Taq, Tth, Tne, Tma, Tli, Pfu, Pwo, Bst, Bca, Sac, Tac, Tfl/Tub,Tru, Mth, Mtb, and Mlep DNA polymerases and the like.

The composition of the 5× buffer provided can also be varied, forexample, by use of other buffers such as sulfate containing buffers oracetate based buffers that have been used for cDNA synthesis. It will beapparent to those skilled in the art that different formulations can beoptimized for different applications.

As described supra, amplification of RNA sequences by PCR can beaccomplished by a two step or a one step protocol. Mastermixformulations can be prepared for use in one step RT PCR by changing theprimers and by inclusion of an appropriate thermostable DNA polymerasesuch as Taq DNA polymerase. A variety of formulations have beendescribed for One-step RT PCR, however, in all cases the buffers andenzymes are kept separately and are only mixed immediately prior toreverse transcription reaction. According to the methods of theinvention, the reverse transcriptase, Taq DNA polymerase and buffers,dNTP's, cofactors and all other components for one step RT PCR can bemixed together in a variety of different concentrations to provide aready to use mastermix.

EXAMPLES Methods

RNA Isolation and Purification:

Total RNA from HeLa S3 cells was isolated using Trizol (Invitrogen,Carlsbad, Calif.) according to manufacturer's recommendation. Followingtreatment with RNase-free DNAse to degrade residual genomic DNAcontamination, the RNA was purified by a silica spin cartridge, RNeasy,(Qiagen), and quantified by optical absorbance at 260 nm.

cDNA Synthesis:

First-strand cDNA synthesis was carried out using supplied components ofthe SuperScript First-Strand Synthesis System for RT-PCR, (Invitrogen).In certain experiments, M-MLV RT, diluted to 50 U/μl in enzyme storagebuffer, was substituted for SuperScript II RT. Primers for cDNAsynthesis, hexamer, octamer, or oligo(dT)₂₀ were from Oligos Etc.Reactions (20 μl volumes) were assembled on ice with all requiredcomponents including: buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl); 0.5 mMeach dNTP, 5 mM magnesium chloride, 10 mM dithiothreitol (DTT), 20 unitsRNase inhibitor protein, 50 units of reverse transcriptase, varyingamounts of HeLa cell total RNA, and primer(s) as indicated in eachexample. First-strand reactions were incubated 5-10 mM at 25° C.,followed by 30 minutes at 42° C. Following first-strand synthesis,reactions were heat-killed at 85° C. for 5 min., diluted in TE bufferand stored at 4° C.

Real-Time Quantitative PCR:

Real-time PCR was carried out in 50-μl reaction volumes using theiCycler and iQ SYBR Green Supermix (Bio-Rad Laboratories) according tomanufacturer's recommendation. Target specific mastermixes were preparedwith 300 nM each primer and dispensed as 40-μl volumes into 96-well PCRplates. cDNA sample (10 μl) was added to the appropriate wells and theplate was sealed with a clear heat-seal film (Marsh Bio Products).Reactions were mixed by vortexing then centrifuged briefly to collectcontents in the bottom of each well. qPCRs were incubated for 3 min at95° C. followed by 45 cycles of 95° C., 3 min.; 60 C, 30 s. Fluorescentsignal was collected and analyzed at the 60° C. annealing/extensionstep.

Primer Sequences for qPCR:

Primers used for SYBR Green I real-time PCR were designed using theOLIGO software program (Molecular Biology Insights) or Primer Express(Applied Biosystems). Primer and target sequence information aresummarized in the table 1.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

Example I cDNA Priming Method Influences mRNA Quantification byReal-Time RT-PCR

Varying amounts of random hexamer (25 ng, 50 ng, 100 ng, 200 ng, or 400ng) or oligo(dT)₂₀ (25 nM, 50 nM, 100 nM, 200 nM, 0.5 μM, 1 μM, or 2 μM)or a mixture of 250 ng hexamer and 100 nM oligo(dT)₂₀ were used to primefirst-strand cDNA synthesis from either 200 ng or 200 pg of HeLa celltotal RNA in 2040 volumes as describes above with either SuperScript IIor M-MLV RT. Each reaction was performed in triplicate. After completionof first-strand synthesis, reactions were diluted to 200 μl with 10 mMTris-HCl, pH 8.4, 0.5 mM EDTA and 10 μl aliquots were used as templatefor SYBR Green real-time PCR with primer sets specific for a variety ofhuman transcripts including: replication protein A (RPA),cyclin-dependent kinase inhibitor 1B (Kip1), nuclear cap binding protein(CBP), the 3′-end or 5′-end of RNA-specific adenosine deaminase (ADAR),the 3′-end or 5′-end of adenomatosis polyposis coli (APC),glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, or r18s.Results for average cycle threshold (C_(T)) values obtained for eachqPCR are summarized in Table 2.

Maximal sensitivity (lowest C_(T)) for each specific transcript variedwith the choice and amount of cDNA primer. In general, higherconcentrations of random hexamer resulted in increasing sensitivity ofdetection with optimal results obtained between 200 and 400 ng ofhexamer primer. Results were consistent whether starting with 200 ng or200 pg of total RNA template. These data contradict earlier studies thatemphasized the importance of optimizing the ratio of random primer toRNA template. Furthermore, these results contradict those of Deprez etal., 2002. Anal Biochem. 307:63-69, who found random primer usagegrossly inefficient for quantitative real-time PCR.

For some target sequences, use of oligo(dT)₂₀ resulted in more sensitivedetection than hexamer primer. RPA was detected approximately 2 C_(T)'searlier (˜4 fold) when cDNA was primed with oligo(dT)₂₀ compared tohexamer. Surprisingly, sensitivity of detection for oligo(dT)-primedcDNA was relatively consistent from 50 nM to 2 μM for either SuperScriptII or M-MML RT. Most published cDNA protocols using oligo dT primer, orderivatives of oligo dT, use between 0.5 and 5 μM oligo dT. These datademonstrate that efficient cDNA synthesis can be obtained using muchlower concentrations of primer. Surprisingly, the efficacy of oligodT-primed cDNA with M-MLV was comparable to that obtained withSuperScript II. Comparable qPCR results were obtained for 5′-regions oftemplates requiring reverse transcription of long mRNA sequences such asthe ADAR or APC genes.

For other target sequences (GAPDH, β-actin, 3′-end of ADAR) comparablesensitivities were obtained with either priming method.

Most significantly, use of a mixture of hexamer and oligo dT resulted indetection sensitivities for all target sequences that were comparable tothose obtained using the favored cDNA priming method for any giventarget sequence. Therefore, use of a mixture of oligo dT and randomprimer is likely to result in more consistent performance and uniformcDNA synthesis efficiencies in studies involving genome-wide expressionprofiling.

Example II Lithium Improves cDNA Yield and mRNA Quantification byReal-Time RT-PCR

First-stand cDNA syntheses were performed essentially as described abovein 20 μL reaction volumes using 50 units of MMLV RT and 200 ng or 200 pgof HeLa total RNA. Reactions were primed using a mixture of 100 nMoligo(dT)20, 125 ng random hexamer, and 12 ng random octamer. Controlreactions contained a buffer of 20 mM Tris, pH 8.4, 50 mM KCl. Otherreactions were supplemented with 20 mM LiCl, 20 mM KCl, 20 mM NaCl, or20 mM ammonium sulfate. Each reaction was performed in duplicate. Aftercompletion of first-strand synthesis, reactions were diluted to 200 μlwith 10 mM Tris-HCl, pH 8.4, 0.5 mM EDTA and 10-μl aliquots ( 1/20^(th)of each cDNA reaction) were used as template for SYBR Green real-timePCR with primer sets specific for a variety of human transcriptsincluding: replication protein A (RPA), cyclin-dependent kinaseinhibitor 1B (Kip1), the 3′-end or 5′-end of RNA-specific adenosinedeaminase (ADAR), the 3′-end or 5′-end of adenomatosis polyposis coli(APC), 3′-end or 5′-end of MAP4, β-actin, or r18s. Results for averagecycle threshold (C_(T)) values obtained for each qPCR are summarized inTable 3.

In general, inclusion of lithium ion in the cDNA reaction resulted indetection of cDNA product at lower C_(T)s, indicating a higher cDNAsynthesis efficiency and yield of cDNA product. Addition of 20 mMlithium, potassium, or sodium improved qRT-PCR from low input amounts oftotal RNA (200 pg) as compared to control reactions, with the exceptionof the 3′-end of MAP4. Addition of 20 mM ammonium ion either had littleeffect on C_(T) or resulted in lower cDNA yield as reflected in delayedC_(T)s for RT-PCR product detection. On average, inclusion of lithiumreduced Ct for detection of RT-PCR product from 10 pg of starting totalRNA by 0.6 cycles compared to control reactions and 0.4 cycles comparedto the addition of 20 mM potassium or sodium. Addition of lithiumsignificantly improved the sensitivity of detection for the 3′-end ofthe APC transcript, as this RT-PCR product was not detected in control,or cDNA reactions supplemented with additional potassium or sodium.Similarly, the 5′-end of the ADAR transcript was not detected in controlcDNA reaction. However, RT-PCR product was readily detected inlithium-containing cDNA reactions and was detected approximately 2cycles earlier (˜4 fold higher cDNA yield) as compared to cDNA reactionssupplemented with an equivalent amount of potassium. Two-step qRT-PCRfrom higher amounts of total RNA template (200 ng) also showed improveddetection sensitivities when lithium was included in the cDNA reaction.On the average, the C_(T) for qRT-PCR product from lithium-containingcDNA reactions was 0.3 cycles lower than control reactions or reactionssupplemented with 20 mM potassium, and 0.4 cycles lower than cDNAreactions containing 20 mM sodium. Collectively these data demonstratethat lithium ion improves the efficiency and yield of cDNA synthesis byretroviral reverse transcriptase.

Example III Stability of cDNA Mastermixes

Three c DNA mastermixes were prepared according to the formulationsdescribed above and stored at −20 C. At the indicated times (table 4)the reagents were functionally tested by cDNA synthesis using 1 ug ofHela RNA as template. As control an identically prepared reagent setthat had been stored separately (in buffers recommended in theliterature) were also used to assemble a freshly made cDNA reaction.cDNA synthesis was for initiated at room temperature for 5 minutesfollowed by 30 min incubation at 42 C. The reactions were stopped byheat inactivation at 85 C for 5 min and were diluted 10 fold with TEbuffer. The diluted samples were stored frozen until they were tested byQ-PCR using a set of primers for beta actin. Amplification protocol wasas described in the amplification section above using the IQ SYBR greensupermix and BioRad IQ cycler. Amplifications were performed intriplicates using 1/20^(th) of the cDNA reaction (50 ng of RNA analyzedin each amplification reaction).

1. A method for reverse transcription of one or more nucleic acid molecules comprising incubating one or more nucleic acid templates in a buffer under conditions sufficient to make one or more first nucleic acid molecules complementary to all or a portion of said one or more templates, wherein said buffer comprises: (a) at least one reverse transcriptase; (b) an effective amount of a mixture of random primers, wherein said random primers are present in a concentration of at least about 5 ng/[11; (c) an effective amount of oligo(dT), wherein said oligo(dT) is present in a concentration less than about 211M.
 2. The method according to claim 1 wherein said random primers are present in a concentration of between about 5 ng/111 and about 20 ng/pl
 3. The method according to claim 1 or 2, wherein said oligo(dT) is present in a concentration of between about 25 nM and about 2 1110/1
 4. The method according to any of claims 1-3, wherein said random primers are between 5 and 10 nucleotides long.
 5. The method according to any of claims 1-4, wherein said random primers are random hexamers.
 6. The method according to any of claims 1-5 wherein said oligo(dT) consists essentially of between about 12 and about 25 dT residues.
 7. The method according to any of claims 1-6, wherein said oligo(dT) is oligo(dT)12-1 8 or oligo(dT)₂₀.
 8. The method according to any of claims 1-7, wherein said at least one reverse transcriptase is a viral reverse transcriptase.
 9. The method according to any of claims 1-8, wherein said reverse transcriptase is selected from the group consisting of AMV RT, RSV RT, MMLV RT, HIV RT, EIAV RT, RAV2 RT, TTH DNA polymerase, C. hydrogenoformans DNA polymerase, Superscript II RT, Superscript I RT, Thermoscript RT MMLV and Rnase 1-1″ mutants thereof.
 10. A method for reverse transcription of one or more nucleic acid molecules comprising incubating one or more nucleic acid templates in a buffer under conditions sufficient to make one or more first nucleic acid molecules complementary to all or a portion of said one or more templates, wherein said buffer comprises: (a) at least one reverse transcriptase, (b) one or more primers suitable for priming reverse transcription of said one or more templates, and (c) an effective amount of Li ion.
 11. The method according to claim 10, wherein said reverse transcriptase is a viral reverse transcriptase.
 12. The method according to claim 11, wherein said viral reverse transcriptase is selected from the group consisting of AMV RT, RSV RT, MMLV RT, HIV RT, EIAV RT, RAV2 RT, Superscript 11 RT, Superscript 1 RT, Thermoscript RT MMLV and Rnase if mutants thereof.
 13. The method according to any of claims 10-12, wherein said Li ion is present in a concentration of between about 5 mM to about 200 mM.
 14. The method according to claim 13, wherein said buffer further comprises at least one additional monovalent cation in a concentration between about 20 mM and 200 mM, wherein said monovalent cation is selected from the group consisting of Na, K, and N114, and wherein the total concentration of said Li on and said further monovalent cation is less than or equal to about 200 mM.
 15. The method according to claim 14, wherein said additional monovalent cation is K.
 16. A reagent mixture suitable for use in a reverse transcription reaction of at least one template nucleic acid, comprising: glycerol in a concentration between about 10% and about 40%, a buffer and a reverse transcriptase, wherein said reagent mixture demonstrates prolonged stability when stored at −20° C. and may be used directly for a reverse transcription reaction without adding additional reverse transcriptase.
 17. The mixture according to claim 16, wherein said buffer comprises: a monovalent cation selected from the group consisting of Li Na, K and NH4; a magnesium salt; a reducing agent, nucleoside triphosphates, and at least one non-ionic detergent.
 18. The mixture according to claim 17, wherein said buffer further comprises at least one primer suitable for priming reverse transcription of a template by said reverse transcriptase.
 19. The mixture according to claim 18, wherein said buffer comprises an RNAse inhibitor protein.
 20. The mixture according to any of claims 16-19, wherein said buffer comprises a potassium salt, a magnesium salt, nucleoside triphosphates, DTT, at least one primer suitable for priming reverse transcription of a template by said reverse transcriptase, at least one non-ionic detergent, and an RNAse inhibitor protein.
 21. (canceled) 